There are several disease treatments that could significantly benefit by having cells regenerate after injury or lesion formation, particularly in the central nervous system (CNS), in the immune system and in the gastrointestinal tract. The expression of growth factors and their receptors in the pre-implanted human embryo and maternal reproductive tract indicates that such factors influence growth and differentiation of embryonic cells in an autocrine and paracrine manner. Such growth factors are peptides that variously support survival, proliferation, differentiation, size and function of nerve cells and other lineages of cells. EGF (epidermal growth factor) is the first member found of the EGF family and characterized many years ago (Savage and Cohen, J. Biol. Chem. 247:7609-7611, 1972; and Savage et at., J. Biol. Chem. 247:7612-7621, 1972). Additional members of the EGF family have been found and they include vaccinia virus growth factor (VGF; Ventatesan et al., J. Virol.44:637-646, 1982); myxomavirus growth factor (MGF; Upton et al. J. Virol. 61:1271-1275, 1987), Shope fibroma virus growth factor (SFGF; Chang et at., Mol. Cell. Biol. 7535-540, 1987), amphiregulin (AR; Kimura et al., Nature 348:257-260, 1990), and heparin binding EGF-like factor (HB-EGF; Higashiyama et al., Science 251:936-939, 1991). A common structural feature of these polypeptides is the presence of six cysteine residues that form three disulfide cross links that support a conserved structure that binds to the EGF receptor.
Another member of the EGF family is TGFα and it also binds to the EGF receptor (Todaro et al., Proc. Natl. Acad Sci. USA 775258-5262, 1980). TGFα stimulates the EGF receptor's tyrosine kinase activity and has many cellular functions, such as stimulating a mitogenic response in a wide variety of cell types. TGFcα and EGF mRNAs reach their highest levels and relative abundance (compared to total RNA in the early postnatal period and decrease thereafter, suggesting a role in embryonic development. From a histological perspective, TGFα acts on numerous cell types throughout the body. The active form of TGFα is derived from a larger precursor and contains 50 amino acids. TGFα shares only a 30% structural similarity with the 53-amino acid form of EGF, but including conservation of all six cysteine residues. TGFα is highly conserved among species. For example, the rat and human polypeptides share about 90% homology as compared to a 70% homology as between the rat and human EGF polypeptide. The amino acid sequence of human TGFα is shown in SEQ ID NO: 1. The sequence shows that a family consisting of vaccinia growth factor, amphiregulin precursor, betacellulin precursor, heparin binding EGF-like growth factor, epiregulin (rodent only), HUS 19878 and schwannoma derived growth factor have similar sequence motifs and can be considered as members of the same family based upon their shared cysteine disulfide bond structures.
TGFα is an acid and heat stable polypeptide of about 5.6 kDa molecular weight. It is synthesized as a larger 30-35 kDa molecular weight glycosylated and membrane-bound precursor protein wherein the soluble 5.6 kDa active form is released following specific cleavage by an elastase-like protease. TGFα binds with high affinity in the nanomolar range and induces autophosphorylation to transduce signal with the EGF receptor. TGFα is 50 amino acids in length and has three disulfide bonds to forms its tertiary configuration. All three disulfide bonds must be present for activity. TGFα is stored in precursor form in alpha granules of secretory cells. Moreover, the primary amino acid sequence is highly conserved among various species examined, such as more than 92% homology at the amino acid level as between human and rat TGFα polypeptides.
TGFα has been investigated extensively and has recently been identified as useful for treating a patient with a neurological deficit. This mechanism is thought to stimulate proliferation and migration of neural-origin stem cells to those sites or lesions in a deficit. For example, Parkinson's Disease is characterized by resting tremor, rigidity, inability to initiate movement (akinesia) and slowness of movement (bradykinesia). The motor deficits are associated with progressive degeneration of the dopaminergic innervation to the nucleus accumbens and degeneration of noradrenergic cells of the locus ceruleus and serotonergic neurons of the raphe. Up to 80% of nigral dopamine neurons can be lost before significant motor deficits are manifest. TGFα (full polypeptide) was shown, when infused into rat brains, was useful for the treatment of neurodegenerative disorders. Intracerebroventricular (ICV) or intrastriatal infusions of TGFα induced neuronal stem cell proliferation, but degenerating or damaged or otherwise abnormal cells needed to be present to facilitate migration of the neuronal stem cells to a site of injury on a scale sufficient to impact recovery from an associated neurological deficit. Forebrain neural stem cells, that give rise to migrating progenitor cells that affect treatment and recovery from a neurological deficit disorder, are the migrating cells that affect treatment recovery from a neural deficit disorder (e.g., Parkinson's Disease, Huntington's Disease, Alzheimer's Disease and the like).
Neural stem cells have been found in subependyma throughout the adult rodent CNS (Ray et al. Soc. Neurosci. 22:394.5, 1996) and in the subependyma of adult human forebrain (Kirchenbaum et al., Cerebral Cortex 4576-589, 1994). Thus, the discovery that TGFα stimulates proliferation of neural stem cells and promotes migration to a site of injury or deficit has led to its investigation for the treatment of a neurodegenerative disorder (Alzheimer's Disease, Huntington's Disease and Parkinson's Disease) or CNS traumatic injury (e.g., spinal chord injury), demyelinating disease, CNS inflammatory disease, CNS autoimmune disease (e.g., multiple sclerosis) or CNS ischemic disease (e.g., stroke or brain attack).
A CNS stem cell has the potential to differentiate into neurons, astrocytes and to exhibit replication of itself to provide a resource for self-renewal. Both neurons and glial cells seem to be derived from a common fetal precursor cell. In the vertebrate CNS, multipotential cells have been identified in vitro and in vivo. Certain mitogens, such as TGFα, can cause proliferation of CNS mutipotential cells in vitro and this is the basis for a procedure to harvest such cell, treat them ex vivo to stimulate proliferation in culture and then readminister such cells. Immunohistochemical analysis in the human brain supports the notion that TGFα is widely distributed in neurons and glial cells both during development and during adulthood. In mice genetically altered to lack expression of functioning TGFα, there was a decrease in neural progenitor cell proliferation in forebrain subependymna, providing evidence for TGFα as a proliferative factor for neural progenitor cells.
TGFα is found mainly in various neurons of the CNS during development and in the adult brain in the cerebral neocortex, hippocampus and striatum. It is also found in glial cells, primarily in the cerebral and cerebellar cortex areas. Northern blot analyses showed that TGFα and not EGF (epidermal growth factor) is the most abundant ligand that binds to the EGF receptor in the brain. TGFα mRNA levels were 15-170 times higher than EGF in cerebellum and cerebral cortex. TGFα also appears in germinal centers of the brain during neurogenesis and gliogenesis in the developing brain. In the midbrain, the distribution of TGFα overlaps with tyrosine hydroxylase mRNA and fetal dopaminergic neurons. In culture, TGFα enhanced survival and neurite outgrowth of neonatal rat dorsal ganglion neurons (EGF did not) and survival and differentiation of CNS neurons. TGFα induced proliferation of neural precursor cells of the murine embryonic mesencephalon and further induced a significant increase in the number of astroglia and microglia in fetal rat medial septal cells. TGFα increased glutamic acid decarboxylase activity and decreased choline actetyltransferase activity. Thus, TGFα acted as a general neuronal survival factor affecting both cholinergic and GABAergic neurons. In addition, TGFα is a mitogen for pluripotent brain stem cells. Forebrain subependyma contains nestin positive neural stem cells and their progeny, which are constitutively proliferating progenitor epithelial cells. A “knockout” mouse that was genetically engineered to delete the gene for TGFα showed a reduction in neuronal progenitor cells in the subependyma and a reduction in neuronal progenitors that migrate to the olifactory bulb. In vitro, TGFα promoted dopamine uptake in fetal rat dopaminergic neurons in a dose-dependent and time-dependent manner. TGFα selectively promoted dopaminergic cell survival, enhanced neurite length, branch number and the soma area of tyrosine hydroxylase immunopositive cells. The levels of TGFα were elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's Disease and may represent a compensatory response to neurodegeneration. Further, TGFα prevented a striatal neuronal degeneration in an animal model of Huntington's Disease.
The mucosal epithelium of the intestine is in a continually dynamic state known as “epithelial renewal” in which undifferentiated stem cells from a proliferative crypt zone divide, differentiate and migrate to the luminal surface. Once terminally differentiated, they are sloughed from the tips of the villi. The turnover of the crypt-villus cell population is rapid and occurs every 24-72 hours. Continuous exfoliation of the cells at the villus tip is counterbalanced by ongoing proliferation in the crypt so that net intestinal epithelial mass remains relatively constant. The rapidly-proliferating epithelium of the gastrointestinal tract is extremely sensitive to cytotoxic drugs that are widely used in the chemotherapy of cancer. This “side effect” reduces the tolerated dose of such drugs as it can cause a breakdown of the GI barrier function and septic condition in a patient already immuno-compromised. This can also lead to life-threatening hemorrhage. Therefore, there is a need in the art for the development of products and delivery systems that stimulate the repair and rejuvenation of mucosal epithelium in the gastrointestinal tract to provide benefit to patient receiving chemotherapy and radiation therapy for cancer.
Therefore, there is a need in the art to find improved TGFα mimetic agents that are more economical to produce and are smaller (in terms of molecular weight). The present invention was made to address such a need.